The monofunctional enzyme 10-formyl-tetrahydrofolate synthetase from Clostridium acidici-urici and the recombinant, bifunctional enzyme 10- formyl-tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli have both been purified and crystallized. These enzymes comprise three consecutive catalytic activities from the folate-dependent, single-carbon transfer pathway, which is involved in the biosynthesis of several key building-blocks for cell proliferation, including formylmethionyl-tRNA, purine bases, thymidylate, and several amino acids. No enzyme structure possessing these catalytic activities has been determined. Therefore, high-resolution structure determination of these enzymes will provide an enormous amount of information about the mechanism(s) of folate-dependent carbon-interconversion, and will allow us to design and optimize chemotherapeutic and antibiotic agents against the enzyme active sites. The goals presented in this application are to: 1. Solve the high-resolution, three-dimensional structures of 10-formyl tetrahydrofolate synthetase (THFS) and of 10-formyl tetrahydrofolate cyclohydrolase/dehydrogenase, using data collected at the structural biology facility at the Hutchinson Cancer Research Center. The first enzyme has been crystallized in three different space groups, and the most promising has yielded complete native data sets to 2.5 A resolution, and several isomorphous derivatives to 2.9 A. M.I.R. phasing and subsequent phase refinement using non-crystallographic averaging and solvent flattening is in progress. The second enzyme has recently been crystallized two different space groups, and the most promising has yielded a complete native data sets to 3.0 A resolution. Initial isomorphous derivative screening has identified ethyl mercuric phosphate and K2PtCl4 as a good candidates for isomorphous replacement. 2. Pursue mechanistic studies for both of these enzymes through a combination of substrate and inhibitor complex structures and site-directed mutagenesis, and initial slope kinetic studies. A number of complexes will be examined crystallographically, including: (i) binary substrate complexes with either formate, ATP-Mg++, or tetrahydrofolate, (ii) the N-5', N-10'- platinum adduct of tetrahydrofolate, which is stable to non-enzymatic oxidation and acts as a competitive inhibitor of THFS, and (iii) a quaternary pseudo-michaelis complex using a non-hydrolyzable ATP analogue to prevent turnover.